1. Field of Invention
This invention relates to regulation of plant osmoregulation and transpiration through guard cells. In particular, the present invention relates to the regulation of guard cell opening through the activity of the AtCHX20 promoter. The AtCHX20 promoter serves as a powerful tool to manipulate the opening and closing of guard cells and thus the ability to control water loss and gas exchange of plants.
Although all cells in an organism contain the same genetic make up, each cell expresses a particular subset of genes that give the cell its particular structure and function. Each protein-coding gene is under the control of its own promoter which consists of a distinct DNA sequence. In plants, the ‘promoter’ or regulatory region (usually 1-3 kb in length) is located immediately upstream of the structural gene. Here evidence is provided showing that AtCHX20 is specifically and highly expressed in guard cells. These results demonstrate that AtCHX20 gene is expressed under the control of a guard-cell specific promoter. If so, the guard cell-specific promoter region of AtCHX20 can be used to regulate expression of any gene in guard cells. The promoter region will be a powerful tool when it is used to express genes and proteins that significantly affect the opening and closing of stomatal pores.
2. Description of Prior Art
Most land plants have the ability to regulate gas exchange and transpiration by the opening and closing of the stomatal aperture. The movement of a pair of special epidermal cells or guard cells, controls the size of the stomatal aperture and so regulates the extent of water loss via transpiration and also regulates CO2 uptake into the leaf for photosynthetic carbon fixation.
At the beginning of the day, light stimulates the opening of the stomatal aperture of most plants by increasing solute concentration and decreasing water potential, thus attracting water into the guard cells (for review, see Assmann, 1993; Schroeder et al., 2001; Roelfsema and Hedrich, 2005). The concomitant increase in turgor pressure causes the guard cells to swell and pushes the pair of cells apart, increasing the aperture between the two cells.
At dusk, the aperture size decreases and becomes nearly closed at night, thus reducing transpiration and gas exchange. During drought, the amount of abscisic acid (ABA) reaching the guard cells can increase, triggering the efflux of ions and loss of water and turgor pressure, leading to closure of the stomatal aperture. ABA can also prevent light-induced stomatal opening (Schroeder et al., 2001).
Studies of the osmotic changes driving guard cell movement have focused mainly on the roles of plasma membrane (PM)-associated transporters and signaling elements regulating the transporters (Blatt, 2000; Fan et al., 2004; Roelfsema and Hedrich, 2005). Advances in understanding their activity have been triggered by the ability to patch guard cell PM, to study transport across this membrane, and to analyze mutants.
It has been found that light-induced stomatal opening starts when light activates the PM H+-ATPase causing membrane hyperpolarization. K+ then enters via inward-rectifying channels, and anions enter via predicted H+/Cl− and H+/NO3− symporters. Ion, malate, and sugar accumulation decreases the water potential; thus, water is taken up, increasing turgor pressure.
More recently, several inward-rectifying K+ channels (e.g. KAT1, KAT2, AKT1) in stomatal opening have been identified at the molecular level (for review, see Very and Sentenac, 2003; Fan et al., 2004). Nitrate is one counterion that balances K+ uptake via an H+-coupled NO3− symporter (AtNRT1.1; Guo et al., 2003). Stomatal closing begins when the membrane depolarizes, causing the opening of outward-rectifying K+ channels. Dark-induced depolarization is caused by deactivation of the PM H+ extrusion pump and by opening of anion efflux channels. Loss of K+ and anions leads to a decrease in solute concentration, water efflux, and loss of guard cell turgor. GORK is suggested to be the major outward-rectifying K+ channel (Hosy et al., 2003); however, the molecular identity of PM R-type and S-type anion channels is still unclear. Genetic evidence suggests that the AtMRP5 ABC (ATP-binding cassette) transporter mediates anion efflux (Klein et al., 2003).
Less well understood are the changes of intracellular compartments during guard cell movement. As guard cells increase in volume, the size of vacuoles increases considerably (Louget et al., 1990), indicating that the bulk of solutes entering guard cells accumulate in the large vacuoles (MacRobbie, 1999), which is iso-osmotic with the cytosol. When stomata close, guard cells are filled with numerous relatively small vacuoles. Many vacuolar transporters identified in plant cells are expressed in guard cells according to the Affymetrix 8K GeneChip® results (Leonhardt et al., 2004). Endomembrane compartments, including vacuoles, are acidified by electrogenic H+-pumping vacuolar-type ATPases (V-ATPase) and H+-pumping pyrophosphotases (Sze, 1985; Rea and Poole, 1993). Thus, it is very likely that the vacuolar membrane potential (DCvac) slightly positive inside the lumen relative to the cytosolic side and DpH acidic inside the lumen relative to the cytosol could drive the accumulation of K+ into the lumen via H+/cation antiporters.
Anions, including Cl− and NO3−, were predicted to enter vacuoles via anion-specific channels because these anions rapidly dissipate the membrane potential generated by the V-ATPase of intracellular vesicles (Sze, 1985), although recent evidence showed that NO3− enters vacuoles through a H+-coupled NO3− antiporter (ClC-a) at the vacuolar membrane (De Angeli et al., 2006).
VK channel activity previously characterized to function in K+ release from vacuoles in response to elevated cytosolic Ca2+ (Ward and Schroeder, 1994) is mediated by TPK+/KCO1 (Bihler et al., 2005). FV channels are inhibited by elevated cytosolic Ca2+ and may modulate K+ uptake into vacuoles during stomatal opening (Pei et al., 1999).
Until the disclosure of the present invention, it was not known whether there was a promoter that regulated gene expression in guard cells with high specificity. The applicants herein have identified a promoter which can be used to regulate the opening and closing of these guard cells.